Mrna large scale synthesis and purification

Abstract

Described herein is method for purifying messenger RNA (mRNA) encoding a DNA endonuclease from a sample, the method comprising: (a) loading the sample comprising the mRNA onto a monolithic matrix comprising a poly(dT) or poly(U) nucleic acid molecule linked/coupled to the monolithic matrix under conditions allowing the mRNA to hybridize with the poly(dT) or poly(U) nucleic acid molecule; (b) eluting the mRNA from the monolith matrix after one or more contaminants have been separated from the bound mRNA; and (c) separating the mRNA from dsRNA by adsorption chromatography, thereby resulting in a purified mRNA solution.

1 . A method for purifying messenger RNA (mRNA) encoding SpCas9 from a sample, the method comprising:
 (a) loading the sample comprising the mRNA onto a monolithic matrix comprising a poly(dT) or poly(U) nucleic acid molecule linked/coupled to the monolithic matrix under conditions allowing the mRNA to hybridize with the poly(dT) or poly(U) nucleic acid molecule;   (b) eluting the mRNA from the monolith matrix after one or more contaminants have been separated from the bound mRNA; and   (c) separating the mRNA from dsRNA by adsorption chromatography, thereby resulting in a purified mRNA solution.   
     
     
         2 . A method for separating double stranded RNA (dsRNA) from mRNA encoding SpCas9, the method comprising:
 (a) loading a sample comprising the mRNA with monolithic matrix comprising a poly(dT) or poly(U) nucleic acid molecule linked/coupled to the monolithic matrix under conditions allowing the mRNA to hybridize with the poly(dT) or poly(U) nucleic acid molecule;   (b) eluting the mRNA from the monolith matrix, thereby resulting in a semi-purified mRNA solution; and;   (c) separating the mRNA in the semi-purified mRNA solution from dsRNA by adsorption chromatography, thereby resulting in a purified mRNA solution.   
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein nucleotides in the mRNA are modified. 
     
     
         5 . The method of  claim 4 , wherein the uridines in the mRNA are replaced with N-1-methylpseudouridine, pseudouridine, and/or 5-methoxyuridine. 
     
     
         6 . The method of  claim 1 , wherein the mRNA comprises the nucleotide sequence of SEQ ID NO: 2, and wherein uridines in the mRNA are replaced with N1-methylpseudouridine; 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the one or more contaminants are selected from the group of proteins, unreacted nucleotides, plasmid DNA, CAP analogues, partial transcripts, dsRNA side products and enzymes. 
     
     
         9 . The method of  claim 1 , wherein the mRNA comprises a poly(a) tail and wherein the one or more contaminants lack a poly(a) tail. 
     
     
         10 . The method of  claim 1 , wherein the mRNA is transcribed from a linearized DNA plasmid via an in vitro transcription (IVT) reaction. 
     
     
         11 . The method of  claim 1 , wherein 100% of the uridines in the mRNA are modified and/or replaced with N-1-methylpseudouridine. 
     
     
         12 . A method for producing purified mRNA encoding SpCas9, comprising:
 (a) linearizing a codon optimized DNA plasmid encoding the endonuclease;   (b) subjecting the plasmid of (a) to an IVT reaction in the presence of a modified uridine nucleotide to synthesize mRNA comprising the modified uridine nucleotide;   (c) purifying the mRNA by a method comprising:   (i) loading the sample comprising the mRNA onto a monolithic matrix comprising a poly(dT) or poly(U) nucleic acid linked/coupled to the monolithic matrix such that the mRNA binds the column, wherein the mRNA comprises the nucleotide sequence of SEQ ID NO: 2, and wherein uridines in the mRNA are replaced with N-1-methylpseudouridine;   (ii) eluting the mRNA from the column after one or more contaminants have been separated from the bound mRNA; and   (iii) separating the mRNA of (b) from dsRNA by adsorption chromatography, thereby resulting in an semi-purified mRNA solution;   (iv) separating the mRNA in the semi-purified mRNA solution from dsRNA by adsorption chromatography,   thereby producing a purified mRNA solution.   
     
     
         13 - 14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein adsorption chromatography is reverse phase chromatography. 
     
     
         16 . The method of  claim 15 , where the sample is loaded onto the column for reverse phase chromatography and an elution buffer is about 35% to about 55% Buffer B, optionally about 50% Buffer B, and the remainder comprising Buffer A, wherein Buffer A comprises 0.1 M TEAA and Buffer B comprises 0.1 M TEAA and 25% acetonitrile. 
     
     
         17 . The method of  claim 15 , wherein the flow rate through the column is about 0.5 mL/min-5.0 mL/min, optionally about 3 mL/min. 
     
     
         18 . The method of  claim 15 , wherein the mRNA is loaded onto the column for reverse phase chromatography at a concentration of 0.05-5.00 mg/mL. 
     
     
         19 . The method of  claim 1 , wherein the purified mRNA solution has less than 0.015% dsRNA, or wherein dsRNA is not detectable in the purified mRNA solution. 
     
     
         20 . The method of  claim 1 , wherein the purified mRNA solution is further processed to exchange a buffer. 
     
     
         21 . The method of  claim 20 , wherein the buffer is exchanged by a tangential flow filtration (TFF) system. 
     
     
         22 . The method of  claim 1 , wherein the purified mRNA or mRNA solution has reduced immunogenicity compared to mRNA purified via step (a) and not via adsorption chromatography. 
     
     
         23 . The method of  claim 12 , wherein the purified mRNA solution has reduced immunogenicity compared to mRNA solution purified via step (c)(i) without step (c)(ii). 
     
     
         24 - 25 . (canceled) 
     
     
         26 . A composition of purified mRNA produced by the method of  claim 1 . 
     
     
         27 - 29 . (canceled)